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Abstract:
Translational diffusion in the plasma membrane of individual egg cells from the sea urchin species Paracentrotus lividus has been studied by fluorescence microphotolysis (FM). In order to probe the lipid phase of the membrane, procedures have been worked out by which the fluorescent analog 3,3′-dioctadecyl-oxatricarbocyanine (C18diO) can be incorporated into the membrane. In the unfertilized egg a fraction R = 0.9 of C18diO was mobile having an apparent diffusion coefficient of D = 6.0 × 10−9 cm2 sec−1. Fifteen to twenty-five minutes after fertilization R and D were reduced to 0.8 and 2.7 × 10−9 cm2 sec−1, respectively. In order to study diffusion of membrane proteins, procedures have been worked out by which the cell surface can be labeled with fluorescein-isothiocyanate (FITC). FITC binds to both the plasma membrane and the vitelline layer. Together with the vitelline layer two-thirds of the FITC-fluorescence could be removed from the egg surface. Gel electropherograms of isolated egg cortices showed various protein bands; however, only two of the protein bands were labeled with FITC. In the unfertilized egg a fraction R = 0.9 of the FITC-labeled membrane proteins was mobile having an apparent diffusion coefficient of D = 35 × 10−11 cm2 sem−1. Fifteen to twenty-five minutes after fertilization R and D were reduced to 0.8 and 7.0 × 10−11 cm2 sec−1, respectively. FITC-labeled proteins of the fertilization envelope were immobile. Our studies have shown (i) that the egg surface can be fluorescently labeled without blocking fertilization and early development, (ii) that the plasma membrane of unfertilized eggs is a fluid environment permitting a rapid movement of lipids and proteins, and (iii) that after fertilization a substantial degree of lipid and protein mobility is maintained.
